Acceleration of diabetic wound healing

ABSTRACT

The invention provides compounds, compositions, and methods to improve or accelerate the healing of a wound. In various embodiments, the methods can include the topical treatment of the wound with the enzyme MMP-8, or the topical treatment of the wound with MMP-8 in combination with administration of an MMP-9 inhibitor, to accelerate the healing of the wound.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Nos. 62/052,921, filed Sep. 19, 2014, and62/128,871, filed Mar. 5, 2015, which applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Diabetes affects 340 million people in the world, including 29.1 millionindividuals in the United States. A complication in diabetic patients isthe inability of wounds to heal, which resulted in 73,000 lower-limbamputations in the United States in 2010. The standard treatment fordiabetic foot ulcers includes debridement of the wound, treatment ofinfection with antibiotics, and reducing or eliminating weight pressurefrom the lower extremities. There is currently no pharmacologicaltherapeutic available that accelerates wound healing without significantadverse effects.

In diabetic patients, high blood sugar triggers prolonged chronicinflammation, with concomitant elevated levels of matrixmetalloproteinases (MMPs). The detrimental effect of MMPs in thediseased tissue has been attributed to rapid turnover of potentialgrowth factors, receptors, and the newly formed extracellular matrix,which are essential for wound healing. Hence, wound healing is impairedand delayed in diabetic patients. However, this process is not wellunderstood and the actual instigator MMPs is not known.

MMPs are a family of zinc-dependent endopeptidases that are capable ofdegrading extracellular matrix components and are involved in tissueremodeling and restructuring. MMPs are expressed as zymogens orpro-MMPs. Activation by proteolytic removal of the N-terminal pro-domainis required for their catalytic functions. Active forms of MMPs arehighly regulated by binding of tissue inhibitors of metalloproteinases(TIMPs). MMPs are presumed to play various roles in regulatinginflammatory and repair processes, as well as in wound healing.

In view of the inherent problems associated with chronic wounds andwound healing, there is a need for therapeutic compositions that areeffective for the treatment of such wounds. There is also a need forselective therapies that are effective to enhance and accelerate thehealing process, particularly in diabetic patients.

SUMMARY

Chronic wounds in diabetic patients are a devastating complication ofdiabetes that can lead to amputations or even death. Our work shows thatmatrix metalloproteinase (MMP)-9 contributes to delayed or impairedwound healing and that MMP-8 is involved in repairing the wound. Acombination of a selective inhibitor of MMP-9 (a small molecule) andexogenously applied active recombinant MMP-8 (an enzyme) acceleratesdiabetic wound healing to provide a first-in-class therapy for thehealing of diabetic wounds. The invention therefore providescompositions and methods for the acceleration of diabetic wound healing,for example, using a novel protease-anti-protease combination therapy,as described herein.

The inventors identified active MMP-8 in wounds of non-diabetic anddiabetic mice, and found that MMP-9 was upregulated only in diabeticwounds. Treatment of wounds with a selective MMP-8 inhibitor delayedwound healing in diabetic animals. Selective inhibition of MMP-9 orablation of the MMP-9 gene led to acceleration of wound healing indiabetic mice. Treatment of wounds with the enzyme MMP-8 acceleratedwound healing in both diabetic and non-diabetic animals. These resultsdemonstrate that MMP-8 plays a beneficial role in wound healing and thatMMP-9 makes wounds refractory to healing. Thus, treatment of wounds withthe enzyme MMP-8, with a combination of MMP-8 and an MMP-9 inhibitor, orwith an MMP-9 inhibitor, accelerates wound healing.

Accordingly, the invention provides a therapeutic composition fortreating a wound comprising an effective amount of the enzyme MMP-8 andan effective amount of a selective MMP-9 inhibitor. The selective MMP-9inhibitor can be selective for MMP-9 over MMP-8, wherein the Ki value ofthe inhibitor for MMP-9 is at least 0.5 μM smaller than the Ki value ofthe inhibitor for MMP-8. Additionally, the selective MMP-9 inhibitor canbe selective for MMP-9 over MMP-8, wherein the Ki value of the inhibitorfor MMP-9 is at least 1 μM, at least 2 μM, or at least 5 μM smaller thanthe Ki of the inhibitor for MMP-8. In one embodiment, the MMP-9inhibitor is:

or salt or solvate thereof. In one specific embodiment, the MMP-9inhibitor is:

or salt or solvate thereof.

The invention also provides a therapeutic composition for treating awound comprising an effective amount of a recombinant MMP-8 enzyme incombination with a surfactant, buffer, salt, or a combination thereof.Other components may be included in the composition, as described hereinbelow for MMP-8. Any composition described herein may be formulated incombination with a pharmaceutically acceptable diluent, excipient, orcarrier.

The invention further provides a method of accelerating the healing of awound comprising contacting an open wound of a subject with an effectiveamount of a composition comprising the enzyme MMP-8, therebyaccelerating the healing of the wound. The wound can be contacted withat least about 0.5 μg of MMP-8 per 50 mm² of open wound per day. Themethod can further include administering to a subject having the openwound an effective amount of a selective MMP-9 inhibitor. The selectiveMMP-9 inhibitor can be selective for MMP-9 over MMP-8, wherein the Kivalue of the inhibitor for MMP-9 is at least 0.5 μM smaller than the Kivalue of the inhibitor for MMP-8.

In various embodiments, the MMP-9 inhibitor is:

or salt or solvate thereof. In further embodiments, the selective MMP-9inhibitor is selective for MMP-9 over MMP-8, wherein the Ki value of theinhibitor for MMP-9 is at least 2 μM smaller than the Ki value of theinhibitor for MMP-8. In certain embodiments, the MMP-9 inhibitor is:

or salt or solvate thereof. In one particular embodiment, the MMP-9inhibitor is ND-336.

In some embodiments, at least about 0.05 mg of the MMP-9 inhibitor isadministered to the subject per 50 mm² of open wound per day. In variousembodiments, the wound is a chronic wound. The subject can be a diabeticsubject.

The invention additionally provides a method for decreasing inflammationand increasing angiogenesis in a diabetic wound comprising administeringto a subject having a diabetic wound an effective amount of any one ofthe compositions described above or herein below.

The invention yet further provides a method for reducing the amount ofapoptotic cells in a diabetic wound comprising administering to asubject having a diabetic wound an effective amount of any one of thecompositions described above or herein below.

The invention also provides for the use of a composition comprising theenzyme MMP-8 for accelerating the healing of a wound. The wound can be achronic wound, a diabetic wound, or a combination thereof.

The invention thus provides methods of accelerating the healing processof a wound. The wound can be internal, or the wound can be a wound ofthe integument, such as a skin wound. The methods can includeadministering to a mammal afflicted with a skin wound an effectiveamount of the enzyme MMP-8, or an effective amount of a combination ofMMP-8 and an MMP-9 inhibitor; or a composition thereof.

The invention provides for the use of the compositions described hereinfor use in medical therapy. The medical therapy can include acceleratingthe healing of a wound, such as a wound of an animal, for example, ahuman. The invention also provides for the use of a composition asdescribed herein for the manufacture of a medicament to treat woundconditions in animals. The medicament can include a pharmaceuticallyacceptable diluent, excipient, or carrier. The medicament can beadministered to a subject topically, enterally, or parenterally.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1A-C. Effect of MMP-9 inhibition, topical treatment withexogenously added MMP-8, and combined MMP-9 inhibition and exogenousMMP-8 on diabetic wound healing. A single 8 mm wound was made on thedorsal thorax of db/db mice. * p<0.05, # p<0.01 indicate statisticallysignificant differences in wound closure between the indicated groups.(a) Wound healing after treatment with ND-336 (0.1 mg/wound/day).Mean±SD; n=8/group on days 7, 10, and 14; (b) Wound healing afterexogenously added MMP-8 (1 μg/wound/day). Mean±SD; n=11, 6, and 5 ondays 7, 10, and 14, respectively, for the vehicle group; n=10, 5, and 5on days 7, 10, and 14, respectively, for the MMP-8 group; (c) Woundhealing after treatment with combined ND-336 (0.05 mg/wound/day) andMMP-8 (1 μg/wound/day). Mean±SD; n=5, 6, 4 and 4 for vehicle, ND-336,MMP-8 and ND-336+MMP-8 groups, respectively.

FIG. 2A-D. Effect of MMP-9 inhibition and exogenous MMP-8 on diabeticwound healing. Mice received a single 8 mm excisional wound on thedorsal thorax. Wounds were treated with vehicle, ND-336 (0.05mg/wound/day), MMP-8 (1 μg/wound/day), or ND-336 (0.05 mg/wound/day)plus MMP-8 (1 μg/wound/day). (a) H&E staining for representative woundson day 14. Re-epithelialization is indicated by the black line. Picturesare taken with 10× lens. Scale bars are 50 μm. (b) TUNEL images ofrepresentative wounds on day 14. Arrows point to representativeTUNEL-positive cells (apoptotic cells). Pictures are taken with 10×lens. Scale bars are 50 μm. (c) In-situ zymography with gelatinasefluorogenic substrate DQ-gelatin (green on the left panels) and mergedwith nuclear DNA staining by DAPI (blue on the right panels). Picturesare taken with 40× lens. Scale bars are 50 μm. (d) In-situ zymographywith collagenase fluorogenic substrate DQ-collagen (green on the leftpanels) and merged with nuclear DNA staining by DAPI (blue on the rightpanels). Pictures are taken with 40× lens. Scale bars are 50 μm.

FIG. 3A-F. MMP-9 inhibition and/or exogenous MMP-8 result in decreasedinflammation and increased angiogenesis (line key: circle=vehicle;square=ND-336; triangle=MMP-8; inverted triangle=ND-336+MMP-8). (a)Concentrations of IL-6 as a function of time after wound infliction; *p<0.05, # p<0.01 indicate statistically significant differences in IL-6between vehicle and the indicated group. (b) Area-under-the-curve (AUC)for IL-6 showed that IL-6 levels were significantly reduced upontreatment with ND-336, MMP-8, or combined ND-336 and MMP-8. (c)Concentrations of TGF-β1 as a function of time after wound infliction; *p<0.05, # p<0.01 indicate statistically significant differences inTGF-β1 between vehicle and the indicated group. (d) Area-under-the-curve(AUC) for TGF-β1 showed that TGF-β1 levels were significantly reducedupon treatment with ND-336, MMP-8, or combined ND-336 and MMP-8. (e)Concentrations of VEGF as a function of time after wound infliction; *p<0.05, # p<0.01 indicate statistically significant differences in VEGFbetween vehicle and the indicated group. (f) Area-under-the-curve (AUC)for VEGF showed that VEGF levels were significantly increased upontreatment with ND-336, MMP-8, or combined ND-336 and MMP-8.

FIG. 4A-D. Effect of MMP-9 inhibition on diabetic wound healing. Asingle 8 mm wound was made on the dorsal thorax. Wounds were treatedwith ND-336 (0.1 mg/wound/day or vehicle. (a) H&E staining on day 14.Re-epithelialization is indicated by the black line. Pictures are takenwith 10× lens. Scale bars are 50 μm. (b) TUNEL images of wounds on day14. Arrows point to representative TUNEL-positive cells (apoptoticcells). Pictures are taken with 10× lens. Scale bars are 50 μm. (c)In-situ zymography with gelatinase fluorogenic substrate DQ-gelatin(green on the left panels), and merged with nuclear DNA staining by DAPI(blue on the right panels). Pictures are taken with 40× lens. Scale barsare 50 μm. (d) zymography with collagenase fluorogenic substrateDQ-collagen (green on the left panels) and merged with nuclear DNAstaining by DAPI (blue on the right panels). Pictures are taken with 40×lens. Scale bars are 50 μm.

FIG. 5A-D. Effect of MMP-9 gene ablation on diabetic wound healing.Diabetes was induced in wild-type and MMP-9 knockout mice by treatmentwith streptozotocin (150 mg/kg ip) and confirmed by fasting bloodglucose of >300 mg/dL. Excisional 8 mm wounds were inflicted two weekslater. (a) Wound healing in wild-type and MMP-9 knockoutstreptozotocin-induced diabetic mice. Mean±SD; n=13 and 7 per group ondays 7 and 14, respectively; * p<0.05 indicates statisticallysignificant differences in wound healing between the two indicatedgroups. (b) Representative wound images (all to the same scale) on days0, 7, and 14. (c) H&E staining for representative wounds in wild-typeand MMP-9 knockout streptozotocin-induced diabetic mice.Re-epithelialization is indicated by the black line. Pictures are takenwith 10× lens. Scale bars are 50 μm. (d) TUNEL images of representativewounds on day 7. Arrows point to representative TUNEL-positive cells(apoptotic cells). Pictures are taken with 10× lens. Scale bars are 50μm.

FIG. 6A-B. Topical treatment with exogenously added MMP-8 accelerateswound healing in db/db mice. A single 8-mm punch biopsy lesion on thedorsal thorax was given to mice. Wounds were treated with MMP-8 (1μg/wound/day) or vehicle (saline). (a) H&E staining for representativewounds treated with vehicle or MMP-8. Re-epithelialization is indicatedby the black line. Pictures are taken with 10× lens. Scale bars are 50μm. (b) In-situ zymography with collagenase fluorogenic substrateDQ-collagen (green on the left panels), and merged with nuclear DNAstaining by DAPI (blue on the right panels). Pictures are taken with 40×lens. Scale bars are 50 μm.

FIG. 7A-B. Dose response of ND-336 in diabetic wound healing. (a) Woundhealing in db/db mice treated with ND-336 at 0.05, 0.025, and 0.01mg/wound/day. Mean±SD; n=7/group on days 7, 10, and 14; * p<0.05indicates statistically significant differences in wound healing. (b)Representative wound images (all to the same scale) on days 0, 7, 10,and 14.

FIG. 8. MMP-9 inhibition, exogenous MMP-8 treatment, and combined MMP-9inhibition and exogenous MMP-8 increase angiogenesis as measured byanti-CD31.

DETAILED DESCRIPTION

The inventors have identified and quantified active MMP-8 and MMP-9 in amouse model of diabetic wound healing by the use of a novelinhibitor-tethered resin that binds exclusively to active MMPs, inconjunction with proteomics analyses (Gooyit et al. (2014) ACS Chem Biol9:505-510). Because MMP-9 was observed upregulated only in diabeticwounds and MMP-8 was found in both diabetic and non-diabetic wounds, wehypothesized that MMP-8 was beneficial and that MMP-9 was detrimental indiabetic wound healing. We now report that the use of either an MMP-9inhibitor or application of the active recombinant MMP-8 accelerateswound healing in diabetic mice. Particularly effective is a novel andhighly selective MMP-9 inhibitor of our design (ND-336, compound 1).

We further confirmed the detrimental effect of MMP-9 on diabetic woundhealing by the use of MMP-9-knockout mice. Finally, we document that thecombination of selective small molecule MMP-9 inhibitors plus the activerecombinant MMP-8 enzyme accelerates wound healing even further indiabetic subjects. These therapies provide a first-in-classpharmacological treatment for diabetic wound healing, which therapieshold great promise for recourse in this devastating disease.

Definitions

The following definitions are included to provide a clear and consistentunderstanding of the specification and claims. As used herein, therecited terms have the following meanings. All other terms and phrasesused in this specification have their ordinary meanings as one of skillin the art would understand. Such ordinary meanings may be obtained byreference to technical dictionaries, such as Hawley's Condensed ChemicalDictionary 14th Edition, by R. J. Lewis, John Wiley & Sons, New York,N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with any element described herein, and/or the recitation ofclaim elements or use of “negative” limitations.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrases “one or more” and “at least one” are readily understood by oneof skill in the art, particularly when read in context of its usage. Forexample, the phrase can mean one, two, three, four, five, six, ten, 100,or any upper limit approximately 10, 100, or 1000 times higher than arecited lower limit. For example, one or more substituents on a phenylring refers to one to five, or one to four, for example if the phenylring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percentages, proximate to the recited range that are equivalentin terms of the functionality of the individual ingredient, thecomposition, or the embodiment. The term about can also modify theend-points of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percentages or carbon groups) includes each specific value,integer, decimal, or identity within the range. Any listed range can beeasily recognized as sufficiently describing and enabling the same rangebeing broken down into at least equal halves, thirds, quarters, fifths,or tenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, for use in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

For medical treatment, an “effective amount” refers to an amounteffective to treat a disease, disorder, and/or condition, or to bringabout a recited effect. For example, an effective amount can be anamount effective to reduce the progression or severity of the conditionor symptoms being treated. Determination of a therapeutically effectiveamount is well within the capacity of persons skilled in the art. Theterm “effective amount” is intended to include an amount of a compounddescribed herein, or an amount of a combination of compounds describedherein, e.g., that is effective to treat or prevent a disease ordisorder, or to treat the symptoms of the disease or disorder, in ahost. Thus, an “effective amount” generally means an amount thatprovides the desired effect.

The terms “treating”, “treat” and “treatment” can include (i) inhibitinga disease, pathologic or medical condition or arresting its development;(ii) relieving the disease, pathologic or medical condition; and/or(iii) diminishing symptoms associated with the disease, pathologic ormedical condition. Thus, the terms “treat”, “treatment”, and “treating”can include lowering, lessening, stopping or reversing the progressionor severity of the condition or symptoms being treated. As such, theterm “treatment” can include medical and/or therapeutic administration,as appropriate.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, activity, or group of cells. The inhibition can begreater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example,compared to the growth or progression or activity that occurs in theabsence of the treatment or contacting.

The terms “increase”, “enhance”, and “accelerate” with respect to woundhealing and wound management refer to the faster re-epithelialization ofa wound, optionally in combination with decreased inflammation and/orincreased angiogenesis, in a wound, for example, a diabetic wound,and/or reduced amount of apoptotic cells in a diabetic wound. Theincreasing, enhancing, or accelerating of wound healing can be about 5%greater (with respect to re-epithelialization of the wound), up to about2-fold or 3-fold greater than the healing occurs in the absence of thetreatment or contacting with the compound or composition describedherein. In some embodiments, the increasing, enhancing, or acceleratingof wound healing can be greater than about 5%, greater than about 10%,greater than about 20%, greater than about 25%, greater than about 30%,greater than about 40%, or greater than about 50%, up to about 3-fold or4-fold, for example, compared to the wound healing (e.g.,re-epithelialization, decrease in inflammatory markers, increase inangiogenesis, or reduction of apoptotic cells) that occurs in theabsence of the treatment or contacting. The enhanced healing occurswithout increased rates of malignancy in the subject, such as theincreased malignancy associated with the use of becaplermin.

The compositions and methods described herein can be used for aidingwound management. The term “wound management” refers to therapeuticmethods that induce and/or promote repair of a wound including, but notlimited to, arresting tissue damage such as necrotization, promotingtissue growth and repair, reduction or elimination of an establishedmicrobial infection of the wound and prevention of new or additionalmicrobial infection or colonization. The term can further includereducing or eliminating the sensation of pain attributable to a wound.

A “wound” refers to an injury to the body, including but not limited toan injury from trauma, violence, accident, or surgery. A wound may occurdue to laceration or breaking of a membrane (such as the skin) andusually damage to underlying tissues. A wound may occur in a topicallocation or internally. Chronic wounds may be caused by diseases,including but not limited to diabetes; diseases of internal organs,including but not limited to diseases of the liver, kidneys or lungs;cancer; or any other condition that slows the healing process.

Natural healing occurs in clearly defined stages. Skin wounds of acutenature may heal in 1-3 weeks in a biological process that restores theintegrity and function of the skin and the underlying tissue. Suchwounds may be the result of a scrape, abrasion, cut, graze, incision,tear, or bruise to the skin. If a wound does not heal in 4-12 weeks, itmay be considered chronic. In the case of chronic wounds, the wound maybe attenuated at one of the stages of healing or fail to progressthrough the normal stages of healing. A chronic wound may have beenpresent for a brief period of time, such as a month, or it may have beenpresent for several years. The compositions and methods described hereincan initiate and enhance the healing of a chronic wound, such as achronic skin wound.

The phrase “chronic skin wound” includes, but is not limited to, skinulcers, bed sores, pressure sores, diabetic ulcers and sores, and otherskin disorders. Chronic skin wounds can be any size, shape or depth, andmay appear discolored as compared to normal, healthy skin pigment.Chronic skin wounds can bleed, swell, seep purulent discharge or otherfluid, cause pain or cause movement of the affected area to be difficultor painful. Chronic skin wounds can become infected, producing elevatedbody temperatures, as well as pus or discharge that is milky, yellow,green, or brown in color. The discharge can be odorless or have apungent odor. If infected, chronic skin wounds may be red, tender, orwarm to the touch.

Chronic skin wounds can be caused by diabetes, poor blood supply, lowblood oxygen, by conditions where blood flow is decreased due to lowblood pressure, or by conditions characterized by occluded, blocked ornarrowed blood vessels. A low oxygen supply can be caused by certainblood, heart, and lung diseases, and/or by smoking cigarettes. Chronicskin wounds can also be the result of repeated trauma to the skin, suchas swelling or increased pressure in the tissues, or constant pressureon the wound area. Chronic skin wounds can also be caused by a weakenedor compromised immune system. A weakened or compromised immune systemcan be caused by increasing age, radiation, poor nutrition, and/ormedications, such as anti-cancer medicines or steroids. Chronic skinwounds can also be cause by bacterial, viral or fungal infections, orthe presence of foreign objects.

Therapeutic compositions for use in methods of wound healing and woundmanagement can include one or more surfactants. The surfactant canuseful in cleaning a wound or contributing to bactericidal activity ofthe administered compositions. Suitable surfactants include, but are notlimited to, phospholipids such as lecithin, including soy lecithin, andvarious detergents. In compositions for application to a wound or skinsurface, when a surfactant in included in the composition, thesurfactant selected can be mild and not lead to extensive irritation orpromote further tissue damage to the patient. The surfactant can be anon-natural (e.g., synthetic) surfactant.

A surfactant can be present in the composition at a concentration ofabout 0.1 wt % to about 25 wt %, about 1 wt % to about 20 wt %, about 2wt % to about 20 wt %, or about 5 wt % to about 10 wt %. Suitablenonionic surfactants that can be used in the therapeutic compositionsdescribed herein can include, for example, fatty alcohol ethoxylates(alkylpolyethylene glycols); alkylphenol polyethylene glycols; alkylmercaptan polyethylene glycols; fatty amine ethoxylates(alkylaminopolyethylene glycols); fatty acid ethoxylates(acylpolyethylene glycols); polypropylene glycol ethoxylates(Pluronics); fatty acid alkylolamides (fatty acid amide polyethyleneglycols); alkyl polyglycosides, N-alkyl-N-alkoxypolyhydroxy fatty acidamides, in particular N-methyl-fatty acid glucamide; sucrose esters;sorbitol esters, and esters of sorbitol polyglycol ethers. In oneembodiment, the surfactant is a polypropylene glycol ethoxylate such asthe poloxymer Pluronic F-127 (Poloxamer 407). In other embodiments, thesurfactant(s) can include lecithin, with or without the addition ofPluronic F-127 in about 2 wt % to about 20 wt %, for increasing theviscosity or gelling of the compositions.

Acceleration of Wound Healing.

Non-healing chronic wounds are major complications of diabetes, whichresult in >70,000 annual lower-limb amputations in the United Statesalone. The basis for why the diabetic wound is recalcitrant to healingis not fully understood and there are no therapeutic agents thataccelerate or facilitate its repair. We have identified two active formsof matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds ofdiabetic mice. We thought that MMP-8 might play a role in the body'sresponse to wound healing and that MMP-9 was the pathologicalconsequence of the disease with detrimental effects.

We demonstrate herein that the use of inhibitors of MMP-9, including anovel highly selective inhibitor of MMP-9 (compound ND-336), acceleratediabetic wound healing by lowering inflammation, and by enhancingangiogenesis and re-epithelialization of the wound, hence reversing thepathological condition. The detrimental role of MMP-9 to the pathologyof diabetic wounds was further confirmed by the study of diabeticMMP-9-knockout mice, which exhibited wounds more prone to healing.Furthermore, topical administration of active recombinant MMP-8 alsoaccelerated diabetic wound healing as a consequence of completere-epithelialization, diminished inflammation, and enhancedangiogenesis. The combined topical application of an MMP-9 inhibitor (asmall molecule) and the active recombinant MMP-8 (an enzyme) enhancedhealing even more, a strategy that can be a first-in-class therapeuticin the healing of diabetic wounds.

Synthesis and MMP Inhibition Profile of ND-336.

There are 23 MMPs in humans and their catalytic domains are very similarin structure, thus, the design of inhibitors that are selective for aparticular MMP is extremely challenging. In fact, most inhibitors ofMMPs are zinc chelators, which broadly inhibit many or all MMPs, as wellas the related ADAMs (a disintegrin and metalloproteinase). Over thepast several years we have produced a library of thiirane inhibitors forMMPs (Gooyit et al. (2011) Selective water-soluble gelatinase inhibitorprodrugs. J Med Chem 54(19):6676-6690; Lee et al. (2012)Structure-Activity Relationship for Thiirane-Based GelatinaseInhibitors. ACS Med Chem Lett 3(6):490-495; Testero S A, et al. (2010)Sulfonate-containing thiiranes as selective gelatinase inhibitors. MedChem Lett.), which allowed for identification of specific inhibitors forselective inhibition of enzymes involved in various MMP-mediateddiseases. The thiiranes undergo a reaction catalyzed by the target MMP,resulting in opening of the thiirane ring and generation of thethiolate, which is a tight-binding inhibitor (Forbes et al. (2009)Active site ring-opening of a thiirane moiety and picomolar inhibitionof gelatinases. Chem Biol Drug Des 74(6):527-534). When we identifiedactive MMP-8 and MMP-9 in the diabetic wounds, and hypothesized thatMMP-9 played a detrimental role in the disease, the central criterionfor selectivity of a suitable inhibitor became its ability todifferentiate between MMP-8 and MMP-9, as the latter had to be inhibitedin the presence of active MMP-8. We now report on the discovery ofcompound 1, which meets the requirements for potent inhibition of MMP-9and lack thereof for MMP-8.

The binding constants for ND-336 with seven representative MMPs and tworelated ADAMs are given in Table 1. ND-336 inhibits MMP-2, MMP-9 andMMP-14 in a slow-binding mechanism, with inhibition constant (K_(i))values of 85±1 nM, 150±10 nM, and 120±10 nM, respectively.

TABLE 1 Inhibition profile of ND-336 Enzyme Inhibition type k_(on)(s⁻¹M⁻¹) k_(off) (10³ s⁻¹) Ki MMP-l^(b) 4% inhibition @ 100 μM MMP-2slow-binding  8380 ± 110 0.712 ± 0.006 85 ± 1 nM^(a) MMP-3^(b) 23%inhibition @ 100 μM MMP-7  1% inhibition @ 100 μM MMP-8^(b) linearnon-competitive 7700 ± 100 nM  MMP-9^(b) slow-binding  2360 ± 100 0.352± 0.033 150 ± 10 nM^(a) MMP-14^(b) slow-binding 10800 ± 400 1.33 ± 0.03120 ± 10 nM^(a) ADAM9 31% inhibition @100 μM ADAM10 14% inhibition @100μM ^(a)Calculated from the ratio of k_(off)/k_(on). ^(b)Catalyticdomains.As MMP-2 and MMP-14 are absent in the diabetic wound, the inhibitortargets essentially MMP-9 in this microenvironment. The residence times(the time the drug remains bound to the target; calculated as 1/k_(off))of ND-336 are: 23.4±0.2 min for MMP-2, 47.4±4.4 min for MMP-9, and12.6±0.3 min for MMP-14. For comparison, the residence times of theendogenous protein inhibitor TIMPs are shorter: 6.9 min for MMP-2-TIMP1,10.4 min for MMP-2-TIMP2, 7.9 min for MMP-9-TIMP1, and 6.7 min forMMP-9-TIMP2. That is, ND-336 is better at inhibiting MMP-2 and MMP-9than TIMPs that have evolved for this purpose. ND-336 exhibits marginalto no inhibition of MMP-1, MMP-3, MMP-7, ADAMS and ADAM10, and it poorlyinhibits MMP-8 in a linear noncompetitive manner (K_(i)=7700±100 nM).Combined with the 50-fold lower K_(i) for MMP-9, the exceptionalresidence time contributes substantially to selectivity. The residencetime is an important contributor for effective inhibition of MMP-9. Thisis in contrast to the linear noncompetitive inhibition of MMP-8 byND-336, with a very short residence time and poorer dissociationconstant.

Inhibition of MMP-9 with ND-336 in Diabetic Wound Healing.

ND-336 was evaluated in a mouse model of diabetic wound healing.Excisional wounds were made on the dorsal thorax of diabetic mice andthe wounds were topically treated with either vehicle or ND-336. Woundstreated with ND-336 healed about 2-fold faster, than those given vehicle(FIG. 1a ). As human wounds heal by re-epithelialization, we evaluatedthe wounds with hematoxylin-eosin (H&E) staining for visualization ofthe epithelium. Treatment with ND-336 resulted in almost completere-epithelialization compared to partial re-epithelialization in thevehicle-treated group (FIG. 4a ). As MMP-9 activity is associated withinduction of apoptosis, we evaluated the wounds by the terminaldeoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), whichdetects DNA fragmentation resulting from apoptotic cells. As shown inFIG. 4b , numerous apoptotic cells were found in the vehicle group,while apoptosis was significantly decreased in the ND-336-treated group.

In-situ zymography detects MMP activity in vivo using the fluorogenicsubstrates: DQ-gelatin for gelatinase (MMP-2 and MMP-9) activity andDQ-collagen for collagenase (MMP-1, MMP-8, and MMP-13) activity. Sinceonly active MMP-8 and MMP-9 were identified by our proteomics analysesin diabetic wounds, the gelatinase activity observed by in-situzymography corresponds to MMP-9 activity and the collagenase activity isreflective of MMP-8 activity. Treatment with ND-336 significantlydecreased MMP-9 activity (FIG. 4c left), while MMP-8 activity was notaffected (FIG. 4d left), as expected from the kinetic profile of ND-336(Table 1). Merged images stained with DAPI (blue) indicated comparablenumber of nuclei in the wound tissues treated with vehicle and ND-336(FIGS. 4c right and 4 d right).

Ablation of MMP-9 in Diabetic Wound Healing.

We induced diabetes in MMP-9 knockout mice to confirm the detrimentalrole of MMP-9 in diabetic wound healing. We administered streptozotocin,which destroys insulin-producing beta cells in the pancreas by necrosisand used wild-type mice treated with streptozotocin as the controlgroup. As seen in FIG. 5, the wounds of streptozotocin-treated MMP-9knockout mice healed faster than those of streptozotocin-treatedwild-type and resulted in complete re-epithelialization, as well asdiminished apoptosis. This study confirmed that MMP-9 is involved in thepathology of diabetic wounds. Since enhanced expression of MMP-8 occursin MMP-9 knockout mice, the acceleration of wound healing that weobserve in diabetic MMP-9 knockout mice could be explained by theupregulation of MMP-8 and the absence of MMP-9. Our findings in MMP-9knockout diabetic mice differ from those of Kyriakides et al., whosuggested that MMP-9 is required for normal progression of wound closuresince MMP-9 gene ablation in non-diabetic mice led to delayed woundhealing due to compromised re-epithelialization, attenuated keratinocytewound migration, and reduced clearance of fibrin clots (Kyriakides etal. (2009) Matrix biology: J. Internat. Soc. for Matrix Biology28(2):65-73). However, inflammation and angiogenesis in wounds ofnon-diabetic MMP-9 knockout mice were similar to those in control mice.Others have found that skin inflammation is alleviated in MMP-9 knockoutmice (Purwar et al. (2008) J Invest Dermatol 128(1):59-66) and thatinhibition of MMP-9 with leptomycin B suppressed inflammation inultraviolet B irradiated murine skin (Kobayashi & Shinkai (2005) JInvest Dermatol 124(2):331-337), consistent with our findings.

Effect of MMP-8 in Diabetic Wound Healing.

To test the hypothesis that MMP-8 contributes to the repair in diabeticwound healing, we evaluated the effect of topical application of theprotease MMP-8 to wounds of diabetic mice. We cloned the gene,expressed, and purified active murine recombinant MMP-8 (see Example 2below). The active recombinant MMP-8 was applied topically to the woundsat 10-fold the level found in the wounds (Gooyit et al. (2014) ACS ChemBiol 9:505-510). MMP-8 accelerated wound healing at this level indiabetic mice, with statistical differences observed on days 10 and 14(FIG. 1b ) and resulted in complete re-epithelialization (FIG. 6a ).Increased MMP-8 activity in the MMP-8-treated diabetic mice wasconfirmed by in-situ zymography (FIG. 6b ). This study indicates thatMMP-8 plays a beneficial repair role in diabetic wound healing. Ourfindings are in agreement with those of Gutierrez-Gernandez et al. whofound that non-diabetic mice deficient in MMP-8 have delayed woundhealing ((2007) FASEB J 21(10):2580-2591). Interestingly, MMP-8 knockoutmice have significantly increased levels of MMP-9, due to compensatoryexpression, which contributes to delayed wound healing.

Effect of the Combination of MMP-9 Inhibitor and Exogenously AddedActive Recombinant MMP-8.

As either inhibition of MMP-9 by a small molecule MMP-9 inhibitor (e.g.,ND-336) or topically applied exogenously added active recombinant MMP-8alone accelerated wound healing, we investigated the effect of thecombination therapy. We first determined that 0.05 mg/kg/day of ND-336applied to diabetic wounds was the lowest dose that accelerated woundhealing (FIG. 7). As can be observed in FIG. 1c , the combined treatmentnot only showed significant acceleration of wound healing compared tothe vehicle group on both days 10 and 14, but it also showed significantfaster healing on day 14 than when a single agent was used in thetreatment.

Histological assessment of the wound revealed that the combination ofthe MMP-9 inhibitor and MMP-8 resulted in complete re-epithelializationcompared to either of the two agents by itself or the vehicle (FIG. 2a )and substantial reduction in apoptotic cells relative to the other threegroups (FIG. 2b ) (a 75% reduction). In-situ zymography with DQ-gelatinshowed inhibition of MMP-9 in the ND-336-treated group and in thecombination of ND-336- and MMP-8-treated groups (FIG. 2c , left). WithDQ-collagen, in-situ zymography indicated the presence of MMP-8 in thevehicle- and ND-336-treated groups, while significantly increased MMP-8activity was found in the MMP-8 group and in the group for thecombination of ND-336 and MMP-8 (FIG. 2d left).

MMP-9-Inhibition and Exogenously Added MMP-8 Decrease Inflammation andEnhance Angiogenesis.

Inflammation is necessary for normal wound healing. However, increasedor prolonged inflammation has been shown to delay wound healing innon-diabetic mice. Interleukin-6 (IL-6) plays a crucial role in theinflammatory response in wound repair and it is a proinflammatorycytokine. IL-6 deficient mice display impaired wound healing, which isreversed with administration of IL-6. Delayed wound healing in IL-6knockout mice was accompanied by attenuated leukocyte infiltration,re-epithelialization, angiogenesis, and collagen accumulation.Transforming growth factor-β1 (TGF-β1) is a cytokine that elicitsrecruitment of inflammatory cells during wound healing. TGF-β1 isunregulated during wound healing, indicating that it regulates woundrepair. Immunodeficient TGF-β1 knockout mice show delayed wound healing,with accompanying delays in the inflammatory, proliferation, andmaturation phases of wound healing. TGF-β induces pro-MMP-9 in humanskin and TGF-β1 stimulates the production of MMP-9 in human cornealepithelial cells and in human keratinocytes. Enhanced TGF-β1 signalingaccelerates re-epithelialization.

These findings indicate that IL-6 and TGF-β1 play important roles inwound healing. We, thus, measured the concentrations of IL-6 and TGF-β1by enzyme-linked immunosorbent assay (ELISA) in wounds of diabetic mice.

In vehicle-treated db/db mice, IL-6 was elevated throughout the courseof the study (FIG. 3a ). Treatment with either ND-336 or MMP-8significantly reduced IL-6, and combination of ND-336 and MMP-8decreased IL-6 the most (FIGS. 3a and 3b ). Likewise, treatment withND-336, MMP-8, or combined ND-336 and MMP-8 significantly reduced thelevels of TGF-β1 (FIGS. 3c and 3d ).

Angiogenesis is essential for wound healing, facilitating debris removaland granulation tissue development that facilitate wound closure.Cluster of differentiation 31 (CD31) is found on the surface ofendothelial cells and it is a widely used marker for angiogenesis. Usinganti-CD31 antibodies we found increased angiogenesis in the ND-336,MMP-8, and combined ND-336 plus MMP-8 groups (FIG. 8). Angiogenesis wasquantified using vascular endothelial growth factor (VEGF), whichenhances vascular permeability, promoting formation of new bloodvessels. The levels of VEGF were determined as a function of time afterwound infliction. Treatment with ND-336, MMP-8, or combined ND-336 andMMP-8 increased VEGF concentrations in the wounds compared to vehicle(FIGS. 3e and 3f ). Our results are in agreement with the increased VEGFlevels in human wound fluid (Nissen et al. (1998)Am J Pathol152(6):1445-1452) and in epidermal keratinocytes (Ferrara et al. (2003)Nat Med 9(6):669-676).

In summary, we have shown that MMP-9 inhibitors such as the novel MMP-9inhibitor ND-336 accelerates diabetic wound healing by decreasinginflammation and by enhancing angiogenesis and re-epithelialization ofthe wound, thus, reversing the pathological condition. Topicaladministration of active recombinant MMP-8 accelerated diabetic woundhealing, resulting in complete re-epithelialization, diminishedinflammation, and enhanced angiogenesis. The combination of a selectiveMMP-9 inhibitor with added MMP-8 was the best strategy to acceleratediabetic wound healing and can significantly improve the treatment ofdiabetic wounds. These compositions and methods described herein thusprovide an effective pharmacological treatment for the healing diabeticwounds.

Accordingly, the combination of a selective MMP-9 inhibitor such asND-336 and the enzyme MMP-8 accelerates diabetic wound healing, and inparticular, accelerates re-epithelialization. MMP-8 was found toaccelerate the healing of wounds in both diabetic and non-diabeticsubjects, and both chronic and acute wounds. Additionally, theadministration of a selective MMP-9 inhibitor can further enhance thehealing of a wound exogenously treated with MMP-8.

Compositions and Methods of Treatment

Based on the analysis described herein, it has been discovered thatadministration of MMP-8 accelerates the healing of wounds, in bothdiabetic and non-diabetic subjects. Further administration of aselective MMP-9 inhibitor in conjunction with the administration ofMMP-8 further enhances the healing of a wound. An MMP-9 inhibitor usedin a composition or method with MMP-8 can be a compound of Formula XI:

wherein

-   -   R¹ is (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy,        aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl, aryl(C₁-C₆)alkoxy,        heteroaryl(C₁-C₆)alkoxy, aryl, heteroaryl, hydroxy, SR⁵, NR⁵R⁵,        or absent;    -   R² is CH₂, carbonyl, SO₂, H, or OH;    -   L is CH₂, NR⁵, O, or a direct bond;    -   W¹-W⁶ are each independently C, N, O, S, or absent, and form a 5        or 6 membered aryl, heterocycle, or heteroaryl ring;    -   W^(1′)-W^(6′) are each independently C, N, O, S, or absent, and        form a 5 or 6 membered aryl, heterocycle, or heteroaryl ring;    -   the dashed circles within the rings formed by W¹-W⁶ and        W^(1′)-W^(6′) denote optional double bonds of the rings formed        by W¹-W⁶ and W^(1′)-W^(6′);    -   R³ and R⁴ are each independently hydroxy, (C₁-C₆)alkyl,        (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl,        heteroaryl, carboxy, cyano, nitro, halo, trifluoromethyl,        trifluoromethoxy, SR⁵, SO₂N(R₅)₂, NR⁵R⁵, or COOR⁵;    -   each n is independently 0 to 4;    -   each R⁵ is independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,        (C₆-C₁₀)aroyl, aryl, aryl(C₁-C₆)alkyl, heteroaryl,        heteroaryl(C₁-C₆)alkyl, or a nitrogen protecting group;    -   X is O, S, SO, SO₂, CH₂—O, CH₂—S, CH₂—NR⁵, NR⁵, carbonyl, or a        direct bond;    -   D is S, SO, SO₂, P(O)OH, P(O)O(C₁-C₆)alkyl, P(O(C₁-C₆)alkyl)₂,        C═N—OH, or carbonyl;    -   E is a direct bond, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,        (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, or (C₃-C₈)heterocycle;    -   J is S, O, or NR⁵;    -   G, T, and Q are each independently H, (C₁-C₆)alkyl, or cyano;    -   any alkyl, amino, aryl, heteroaryl, or cycloalkyl is optionally        substituted with 1 to about 5 (C₁-C₆)alkyl, (C₁-C₆)alkoxy, aryl,        heteroaryl, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl, nitro,        halo, amino, or hydroxy groups;    -   or a pharmaceutically acceptable salt thereof. In some        embodiments, when L is CH₂ or O, and R² is CH₂, R¹ is not        (C₁-C₆)alkyl; when L is O and R² is carbonyl, R¹ is not        (C₁-C₆)alkyl; and/or when L is NR⁵, R² is CH₂.

An MMP-9 inhibitor used in a composition or method with MMP-8 can alsobe a compound of Formula XII:

wherein

-   -   X is O, —S—NH—, NW wherein R^(a) is H or (C₁-C₄)alkyl;    -   R¹ is a solubilizing group comprising 5-30 atoms, in addition to        hydrogen, selected from carbon, oxygen, nitrogen, sulfur, and        phosphorus;    -   each R² is independently hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,        (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy,        cyano, nitro, halo, trifluoromethyl, trifluoromethoxy, SR^(z),        SO₂N(R^(z))₂, NR^(z)R^(z), or COOR^(z); wherein each R^(z) is        independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, (C₆-C₁₀)aroyl,        aryl, aryl(C₁-C₆)alkyl, heteroaryl, heteroaryl(C₁-C₆)alkyl, or        optionally a nitrogen protecting group when R^(z) is covalently        bonded to a nitrogen atom; and    -   each n is independently 0, 1, 2, 3, or 4;    -   or a salt or solvate thereof. The compound can have an aqueous        solubility of at least 5 mg/mL.

An MMP-9 inhibitor used in a composition or method with MMP-8 can alsobe a compound of Formula XIII:

wherein R is H, OH, NH₂, NH-amino acid, or —X—(C═O)—R′ where X is O orNH, and R′ is alkyl, aryl, alkylaryl, amino, or alkoxy, where any alkyl,aryl, or amino is optionally substituted.

An MMP-9 inhibitor used in a composition or method with MMP-8 can alsobe a compound of Formula XIV:

wherein

-   -   R¹ is —CH₂—NHR^(a) wherein R^(a) is H or (C₁-C₆)alkanoyl;        —NH—C(═NH)—NH₂; or

-   -   J is S or O;    -   G, T, and Q are each independently H, (C₁-C₆)alkyl, or —CN;    -   each R² is independently H, OH, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,        (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, aryl, heteroaryl, carboxy,        cyano, nitro, halo, trifluoromethyl, trifluoromethoxy, SR^(z),        SO₂N(R^(z))₂, NR^(z)R^(z), or COOR^(z); wherein each R^(z) is        independently H, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, (C₆-C₁₀)aroyl,        aryl, aryl(C₁-C₆)alkyl, heteroaryl, heteroaryl(C₁-C₆)alkyl, or        optionally a nitrogen protecting group when R^(z) is covalently        bonded to a nitrogen atom; and    -   each n is independently 0, 1, 2, 3, or 4;        or a salt or solvate thereof.

Preferably, the MMP-9 inhibitor of Formula XI-XIV is selective for MMP-9over MMP-8, wherein the Ki value of the inhibitor for MMP-9 is at least0.5 μM smaller than the Ki value of the inhibitor for MMP-8.

Table 1 provides several specific MMP-9 inhibitors that can beadministered in conjunction with the compositions and methods of theinvention, such as the exogenous application of MMP-8 to a wound. MMP-9inhibitors that are selective for MMP-9 relative to MMP-8 (e.g., havinga difference in Ki of greater than about 0.5 μM, greater than about 1.0μM, greater than about 1.5 μM, greater than about 2 μM, greater thanabout 5 μM, or greater than about 7 μM; selective for MMP-9) areparticularly effective.

TABLE 1 Thiiranes with MMP-9 Selectivity Relative to MMP-8. NameStructure MMP-9 K_(i) MMP-8 SB-3CT

K_(i) = 0.40 ± 0.15 μM slow-binding residence time = 13.4 min K_(i) =2.1 ± 0.4 μM linear non-competitive ND-322

K_(i) = 0.40 ± 0.15 μM slow-binding residence time = 31.4 min K_(i) =2.6 ± 0.4 μM linear non-competitive ND-364

K_(i) = 0.13 ± 0.01 μM non-competitive K_(i) = 3.4 ± 0.4 μMnon-competitive p-OH SB-3CT

K_(i) = 0.16 ± 0.02 μM slow-binding residence time = 24.5 min K_(i) =2.6 ± 0.3 μM non-competitive ND-336

K_(i) = 0.15 ± 0.01 μM slow-binding residence time = 47.3 min K_(i) =7.7 ± 0.1 μM non-competitive ND-380

K_(i) = 0.18 ± 0.03 μM slow-binding residence time = 19.6 min K_(i) = 13± 2 μM non-competitive ND-394

K_(i) = 0.86 ± 0.11 μM slow-binding residence time = 25.6 min K_(i) = 15± 3 μM non-competitive ND-364

K_(i) = 0.093 ± 0.008 μM slow-binding residence time = 47.0 min K_(i) =0.73 ± 0.05 μM competitive ND-395

K_(i) = 0.93 ± 0.02 μM slow-binding residence time = 119 min K_(i) = 11± 3 μM JNMS-38

K_(i) = 0.005 ± 0.001 μM slow-binding residence time = 152 min K_(i) =1.4 ± 0.48 μM linear competitiveAdditional MMP-9 inhibitors, including MMP-9 inhibitors that areselective for MMP-9 relative to MMP-8, are further described in U.S.Pat. No. 6,703,415 (Mobashery et al.); U.S. Pat. No. 7,114,917(Mobashery et al.); U.S. Pat. No. 7,928,127 (Lee et al.), U.S. Pat. No.8,093,287 (Lee et al.); and U.S. Pat. No. 8,937,151 (Chang et al.); U.S.Patent Publication No. 2013/0064878 (Chang et al.); and InternationalPublication No. WO 2015/127302 (Chang et al.), which patent documentsare incorporated herein by reference, and wherein the compound formulasand individual compounds are individually incorporated herein byreference.

Preferably, the selective MMP-9 inhibitor has a long residence time(e.g., greater than 10 minutes, greater than 15 minutes, greater than 20minutes, greater than 30 minutes, greater than 45 minutes, or greaterthan 100 minutes) with respect to MMP-9. The selective MMP-9 inhibitoryis also preferably a slow-binding inhibitor of MMP-9, and/or acompetitive inhibitor of MMP-8 (i.e., very little or no residence timewith respect to MMP-8).

Accordingly, the invention provides a pharmaceutical compositioncomprising MMP-8 and a pharmaceutically acceptable diluent, carrier, orexcipient. The MMP-8 can be a recombinant MMP-8. The composition thatincludes MMP-8 can be used to treat a wound in a mammal by topicallyapplying the composition to the wound. The amount of MMP-8 can be atleast about 0.5 μg per 50 mm² of open wound per day. Greater amounts ofMMP-8 can also be used, such as 1 μg per 50 mm² of open wound, 1.5 μgper 50 mm² of open wound, 2 μg per 50 mm² of open wound, 2.5 μg per 50mm² of open wound, or 5 μg per 50 mm² of open wound, or even greateramounts. Such doses can be applied once per day, or multiple times perday. The dose can also be divided and applied at various timesthroughout the day.

A subject that has a wound can be administered an MMP-8 composition incombination with administration of an MMP-9 inhibitor, orally,intraperitoneally, or topically. The MMP-9 inhibitor can be a selectiveMMP-9 inhibitor (e.g., selective for MMP-9 over MMP-8). The amount ofthe MMP-9 inhibitor can be at least about 0.05 mg per 50 mm² of openwound per day. Greater amounts of the MMP-9 inhibitor can also be used,such as 0.1 mg per 50 mm² of open wound, 0.2 mg per 50 mm² of openwound, 0.25 mg per 50 mm² of open wound, 0.5 mg per 50 mm² of openwound, or 1 mg per 50 mm² of open wound, or even greater amounts. Suchdoses can be applied once per day, or multiple times per day. The dosecan also be divided and applied at various times throughout the day.

In one embodiment, the MMP-9 inhibitor is SB-3CT. In another embodiment,the MMP-9 inhibitor is ND-322. In another embodiment, the MMP-9inhibitor is ND-364. In another embodiment, the MMP-9 inhibitor is p-OHSB-3CT. In another embodiment, the MMP-9 inhibitor is ND-336. In anotherembodiment, the MMP-9 inhibitor is ND-380. In another embodiment, theMMP-9 inhibitor is ND-394. In another embodiment, the MMP-9 inhibitor isND-364. In another embodiment, the MMP-9 inhibitor is ND-395. In yetanother embodiment, the MMP-9 inhibitor is JNMS-38. In furtherembodiments, the administration of one MMP-9 inhibitor can beaccompanied by the administration of one or more other of theaforementioned MMP-9 inhibitors.

Pharmaceutical Formulations

The compounds (e.g., MMP-8 and/or inhibitors of MMP-9) described hereincan be used to prepare therapeutic pharmaceutical compositions, forexample, by combining the compounds with a pharmaceutically acceptablediluent, excipient, or carrier. The compounds may be added to a carrierin the form of a salt or solvate. For example, in cases where compoundsare sufficiently basic or acidic to form stable nontoxic acid or basesalts, administration of the compounds as salts may be appropriate.Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids that form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, andβ-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, halide, sulfate, nitrate, bicarbonate, andcarbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid to provide aphysiologically acceptable ionic compound. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for example,calcium) salts of carboxylic acids can also be prepared by analogousmethods.

The compounds of the formulas described herein can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient, in a variety of forms. The forms can be specificallyadapted to a chosen route of administration, e.g., oral or parenteraladministration, by intravenous, intramuscular, topical or subcutaneousroutes.

The compounds described herein may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. For oral administration,compounds can be enclosed in hard or soft shell gelatin capsules,compressed into tablets, or incorporated directly into the food of apatient's diet. Compounds may also be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations typically contain at least 0.1% ofactive compound. The percentage of the compositions and preparations canvary and may conveniently be from about 0.5% to about 60%, about 1% toabout 25%, or about 2% to about 10%, of the weight of a given unitdosage form. The amount of active compound in such therapeuticallyuseful compositions can be such that an effective dosage level can beobtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations anddevices.

The active compound may be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can be prepared in glycerol, liquidpolyethylene glycols, triacetin, or mixtures thereof, or in apharmaceutically acceptable oil. Under ordinary conditions of storageand use, preparations may contain a preservative to prevent the growthof microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the active ingredient adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. The ultimate dosage form should besterile, fluid and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions, or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and/or antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, optionally followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation can includevacuum drying and freeze drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thesolution.

For topical administration, compounds may be applied in pure form, e.g.,when they are liquids. However, it will generally be desirable toadminister the active agent to the skin as a composition or formulation,for example, in combination with a dermatologically acceptable carrier,which may be a solid, a liquid, a gel, or the like.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Useful liquidcarriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, orwater-alcohol/glycol blends, in which a compound can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using a pump-type or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of dermatological compositions for delivering active agents tothe skin are known to the art; for example, see U.S. Pat. No. 4,992,478(Geria), U.S. Pat. No. 4,820,508 (Wortzman), U.S. Pat. No. 4,608,392(Jacquet et al.), and U.S. Pat. No. 4,559,157 (Smith et al.). Suchdermatological compositions can be used in combinations with thecompounds described herein where an ingredient of such compositions canoptionally be replaced by a compound described herein, or a compounddescribed herein can be added to the composition

Useful dosages of the compounds, enzymes, or compositions describedherein can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949 (Borch et al.). The amountof a compound, or an active salt or derivative thereof, required for usein treatment will vary not only with the particular compound or saltselected but also with the route of administration, the nature of thecondition being treated, and the age and condition of the patient, andwill be ultimately at the discretion of an attendant physician orclinician.

The compound can be conveniently administered in a unit dosage form, forexample, containing 5 to 1000 mg/m², conveniently 10 to 750 mg/m², mostconveniently, 50 to 500 mg/m² of active ingredient per unit dosage form.The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The compositions can be used to treat a wound in an animal, such as amammal. A mammal includes a primate, human, rodent, canine, feline,bovine, ovine, equine, swine, caprine, bovine and the like.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES

Abbreviations.

ADAM, a disintegrin and metalloproteinase; AUC, area-under-the-curve;Boc, t-Butoxycarbonyl; m-CPBA, meta-chloroperbenzoic acid; DMF,dimethylformamide; DMSO, dimethyl sulfoxide; ESI, electrosprayionization; IL-6, interleukin-6; MMP, matrix metalloproteinase; MOCAc,(7-methoxycoumarin-4-yl)acetyl; PBS, phosphate buffered saline; THF,tetrahydrofuran; TIMP, tissue inhibitor of metalloproteinase; TGF-β1,transforming growth factor 131; TLC, thin-layer chromatography; TUNEL,terminal deoxynucleotidyl transferase-mediated DUTP-nick-end labeling;UPLC, ultra-performance liquid chromatography; VEGF, vascularendothelial growth factor.

Example 1. Synthesis and Analysis of MMP Inhibitors

Synthesis of ND-336. The synthesis of ND-336 is shown in Scheme 1. Thereaction of 4-mercaptophenol (2) with allyl bromide (1) producedcompound 3, which was allowed to react with 4-fluorobenzonitrile toafford diphenyl ether 4 in good yields (94% and 82% for the first andsecond steps, respectively). Subsequent reduction of the nitrile withLiAlH₄, followed by Boc-protection of the resultant amine yieldedcompound 5, which was oxidized to the corresponding oxirane 6. Thereaction of 6 with thiourea produced the Boc-protected thiirane 7. Afterthe final acid Boc-deprotection, the desired ND-336 was in hand as theHCl salt.

Other MMP-9 inhibitors can be prepared by the methods described in U.S.Pat. No. 6,703,415 (Mobashery et al.); U.S. Pat. No. 7,114,917(Mobashery et al.); U.S. Pat. No. 7,928,127 (Lee et al.), U.S. Pat. No.8,093,287 (Lee et al.); and U.S. Pat. No. 8,937,151 (Chang et al.); U.S.Patent Publication No. 2013/0064878 (Chang et al.); and InternationalPublication No. WO 2015/127302 (Chang et al.); each incorporated hereinby reference.

Chemistry.

All reactions were performed under nitrogen atmosphere, unless otherwisenoted. ¹H and ¹³C NMR spectra were recorded on Varian INOVA-500 orVarian UnityPlus 300 spectrometer (Varian Inc., Palo Alto, Calif., USA),Bruker AVANCE III HD 500 or Bruker AVANCE III HD 400 (BrukerCorporation, Billerica, Mass., USA). TLC silica gel 60 F₂₅₄ aluminumsheets (EMD Millipore Corporation, Billerica, Mass., USA) were used forthin-layer chromatography. Flash chromatography was performed with anautomated chromatograph system: Combiflash RF 200i UV/Vis (TeledyneIsco, Lincoln, Nebr., USA). High-resolution mass spectra were obtainedby ESI ionization using a BrukermicrOTOF/Q2 mass spectrometer(BrukerDaltonik, Bremen, Germany). Purity of the prepared compounds wasin general >95%, as confirmed by UPLC. Conditions are detailed in theUPLC section. 4-(Allylthio)phenol (3) was prepared as previouslydescribed (Ikejiri M, et al. (2005) J Biol Chem 280(40):33992-34002;Goux et al. (1994) Tetrahedron 50(34):10321-10330).

4-(4-(Allylthio)phenoxy)benzonitrile (4)

A mixture of 3 (1.45 g, 8.72 mmol), 4-fluorobenzonitrile (1.01 g, 8.38mmol), and Cs₂CO₃ (4.26 g, 13.1 mmol) in DMF (50 mL) was heated at 100°C. for 3.5 h. After the addition of saturated aqueous LiBr (250 mL), themixture was extracted with hexanes/EtOAc (9:1). The combined organiclayers were washed with water and brine, dried over anhydrous Na₂SO₄,and concentrated under reduced pressure. The resultant residue waspurified by silica gel chromatography (hexanes/EtOAc, 97:3) to give 4(1.84 g, 82%) as an oil. ¹H NMR (300 MHz, CDCl₃) δ 7.77-7.52 (m, 2H),7.49-7.30 (m, 2H), 7.14-6.82 (m, 4H), 5.87 (ddt, J=16.9, 10.0, 6.9 Hz,1H), 5.29-4.93 (m, 2H), 3.53 (dt, J=6.9, 1.1 Hz, 2H). ¹³C NMR (126 MHz,CDCl₃) δ 161.6, 153.9, 134.4, 133.7, 132.7, 132.4, 121.0, 119.0, 118.2,118.1, 106.3, 38.2. HRMS (ESI⁺, m/z): calcd for C₁₆H₁₄NO [M+H]⁺,268.0791. found, 268.0799.

t-Butyl 4-(4-(allylthio)phenoxy)benzylcarbamate (5)

A solution of compound 4 (4.98 g, 18.63 mmol) in THF (78 mL) was addeddropwise to LiAlH₄ (2.12 g, 55.89 mmol) in THF (78 mL) at 0° C. over aperiod of 30 min. The ice-bath was removed and the reaction mixture wasstirred at room temperature for 1.5 h at which point the TLC showed thereaction to be complete. The solution was cooled again in ice-watertemperature and quenched carefully with the dropwise addition of 2.4 mLwater, 2.4 mL 15% aqueous NaOH, and 7.2 mL water. The solution wasgradually warmed to room temperature and stirred for 30 min, filteredthrough a celite pad, extracted with diethyl ether and EtOAc. Thecombined organic layer was washed with water and brine, and the solutionwas dried over anhydrous Na₂SO₄. The solvent was evaporated underreduced pressure to give the crude primary amine, which was useddirectly in the next step.

To a mixture of amine (4.3 g, 15.84 mmol) and (Boc)₂O (5.2 g, 23.77mmol) in MeOH/CH₂C₁₂ (3:2, 150 mL), was added a catalytic amount ofiodine (402 mg, 1.58 mmol, 10 mol %). After stirring the reactionmixture for 24 h at room temperature, the solvent was evaporated invacuo, and EtOAc was added. The solution was washed with 5% aqueousNa₂S₂O₃ and saturated NaHCO₃, and dried over anhydrous Na₂SO₄. Thesolvent was evaporated in vacuo, and the residue was purified by silicagel chromatography (hexanes/EtOAc, 95:5) to afford compound 4 (3.21 g,55% in two steps). ¹H NMR (300 MHz, CDCl₃) δ 7.38-7.32 (m, 2H),7.31-7.25 (m, 2H), 6.99-6.90 (m, 4H), 6.17-6.09 (m, 1H), 5.08-5.03 (m,2H), 4.85 (s, b, 1H), 4.29 (d, J=3.0 Hz, 2H), 3.49-3.47 (m, 2H), 1.46(s, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 156.8, 156.4, 156.1, 134.0, 133.2,131.4, 131.2, 129.2, 127.2, 119.3, 117.7, 79.7, 44.4, 38.8, 28.6. HRMS(ESI⁺, m/z): calcd for C₂₁H₂₅NNaO₃S [M+Na]⁺, 394.1447. found, 394.1472.

t-Butyl 4-(4-((oxiran-2-ylmethyl)sulfonyl)phenoxy)benzylcarbamate (6)

m-CPBA (2.03 g, 11.8 mmol) was added in batches to a solution of 5 (0.88g, 2.36 mmol) in CH₂Cl₂ (8 mL) immersed in an ice-water bath. Aftercompletion of the addition, the ice-water bath was removed and thesolution was stirred at room temperature for 3 d. Another batch ofm-CPBA (1.02 g, 5.89 mmol) was added, and the mixture was stirred atroom temperature for an additional 5 d. The suspension was filtered, andthe filtrate was diluted with CH₂Cl₂ and washed with 10% aqueous sodiumthiosulfate, followed by saturated NaHCO₃ and brine. The organic layerwas dried over anhydrous Na₂SO₄, the suspension was filtered, and thesolution was concentrated in vacuo. The product was purified by silicagel chromatography (hexanes/EtOAc, 2:1 to 1:1) to yield 6 (0.36 g, 36%).¹H NMR (400 MHz, CDCl₃) δ 7.91-7.65 (m, 2H), 7.27-7.25 (m, 2H),7.06-6.95 (m, 4H), 5.22 (s, b, 1H), 4.24 (d, J=5.1 Hz, 2H), 3.44-3.02(m, 3H), 2.72 (dd, J=8.0, 2.0 Hz, 1H), 2.39 (dd, J=4.8, 2.0 Hz, 1H),1.39 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 163.0, 154.1, 136.6, 130.7,129.5, 123.2, 120.7, 118.0, 117.8, 79.7, 59.8, 46.04, 45.99, 44.1, 28.6.HRMS (ESI⁺, m/z): calcd for C₂₁H₂₅NNaO₆S [M+Na]⁺, 442.1295. found,442.1292.

t-Butyl 4-(4-((thiiran-2-ylmethyl)sulfonyl)phenoxy)benzylcarbamate (7)

Thiourea (55.3 mg, 0.73 mmol) was added to a solution of compound 6(138.4 mg, 0.33 mmol) in MeOH/CH₂Cl₂ (1:1, 3 mL), and the resultingmixture was stirred at room temperature for 24 h. The solvent wasremoved under reduced pressure and the residue was partitioned betweenCH₂Cl₂ and water. The organic layer was washed with water and brine,dried over anhydrous Na₂SO₄, and filtered. Evaporation of the solventgave the crude product, which was purified by silica gel chromatography(83.4 mg, 58%). ¹H NMR (300 MHz, CDCl₃) δ 7.85 (d, J=8.7 Hz, 2H), 7.33(d, J=8.7 Hz, 2H), 7.13-6.97 (m, 4H), 4.95 (s, b, 1H), 4.33 (d, J=5.9Hz, 2H), 3.51 (dd, J=14.1, 5.7 Hz, 1H), 3.17 (dd, J=14.1, 7.8 Hz, 1H),3.11-2.98 (m, 1H), 2.53 (dd, J=6.3, 1.6 Hz, 1H), 2.15 (dd, J=5.1, 1.6Hz, 1H), 1.46 (s, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 163.1, 156.0, 154.0,136.3, 132.1, 130.8, 129.4, 120.7, 117.8, 79.8, 62.8, 44.1, 28.5, 26.2,24.4. HRMS (ESI⁺, m/z): calcd for C₂₁H₂₅NNaO₅S₂ [M+Na]⁺, 458.1066.found, 458.1089.

(4-(4-((Thiiran-2-ylmethyl)sulfonyl)phenoxy)phenyl)methanamine HCl salt(1)

HCl (0.7 mL, 4 N in 1,4-dioxane) was added to a solution of thiirane 7(61.0 mg, 0.14 mmol) in CH₂Cl₂/EtOAc (1:1, 4 mL). After stirring at roomtemperature for 24 h, the mixture was concentrated under reducedpressure. The resulting crude compound was triturated with diethylether, and the product was obtained by filtration (51.0 mg, 98%). ¹H NMR(300 MHz, CD₃OD) δ 7.93 (d, J=8.6 Hz, 2H), 7.58 (d, J=8.6 Hz, 2H), 7.19(d, d, J=8.4 Hz, 4H), 4.16 (s, 2H), 3.57-3.43 (m, 2H), 3.11-2.99 (m,1H), 2.52 (dd, J=6.3, 1.4 Hz, 1H), 2.14 (dd, J=5.1, 1.4 Hz, 1H). ¹³C NMR(75 MHz, CD₃OD) δ 162.5, 156.2, 133.0, 131.3, 131.1, 130.0, 120.7,118.2, 62.0, 42.6, 25.8, 23.0. HRMS (ESI⁺, m/z): calcd for C₁₆H₁₈NO₃S₂[M+H]⁺, 336.0723. found, 336.0709. The purity of ND-336 was >95%, asdetermined by UPLC with UV detection.

Water-Solubility Determination.

A saturated aqueous solution of ND-336 was prepared and the solution wasfiltered. A 100-fold dilution of the filtrate was analyzed by UPLC withUV detection (see conditions below) using peak area and linearregression parameters calculated from a calibration curve. Thecalibration curve was prepared using known concentrations of ND-336 inacetonitrile. The assay was linear from 0.5 to 100 μg/mL with R² valueof 0.999. The water solubility of ND-336 is 4.90±0.06 mg/mL.

Ultra Performance Liquid Chromatography (UPLC).

A Waters Acquity UPLC System (Waters Corporation, Milford, Mass., USA)equipped with a binary solvent manager, an autosampler, a column heater,and a photodiode array detector was used to test the purity and watersolubility of ND-336. An Acquity UPLC® HSS C18 column (1.8 μm, 2.1×100mm, Waters Corporation, Milford, Mass., USA) was used. The mobile phaseconsisted of water (A) and acetonitrile (B) elution at 0.5 mL/min with85% A, 15% B for 2 min, followed by a 5-min linear gradient to 5% A, 95%B, then 5% A, 95% B for 2 min, followed by a 1-min linear gradient backto 85% A, 15% B and then 85% A, 15% B for 1 min. ND-336 was detected byUV at 245 nm. The retention time was 4.33 min.

Example 2. Acceleration of Diabetic Wound Healing Using a NovelProtease-Anti-Protease Combination Therapy

Enzyme Inhibition Studies.

Human recombinant active MMP-2 and MMP-7, and the catalytic domains ofMMP-3 and MMP-14/MT1-MMP were purchased from EMD Chemicals, Inc. (SanDiego, Calif., USA); human recombinant catalytic domains of MMP-1,MMP-8, and MMP-9 were purchased from Enzo Life Sciences, Inc.(Farmingdale, N.Y., USA); human recombinant active ADAM9 and ADAM10 werepurchased from R&D Systems (Minneapolis, Minn., USA). Fluorogenicsubstrates MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH₂ (for MMP-2,MMP-7, MMP-9 and MMP-14) andMOCAc-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH₂ (for MMP-3) werepurchased from Peptides International (Louisville, Ky., USA);Mca-KPLGL-Dpa-AR-NH₂ (for MMP-1, MMP-8 and ADAM10) andMca-PLAQAV-Dpa-RSSSR-NH₂ (for ADAM9) were purchased from R&D Systems(Minneapolis, Minn., USA). The K_(m) values for MMP-2, MMP-9 and MMP-14were the same as previously reported by Gooyit et al. ((2013) J. Med.Chem. 56(20):8139-8150). Inhibitor stock solutions (10 mM) were preparedfreshly in DMSO before enzyme inhibition assays. We followed the samemethodology for enzyme inhibition studies as reported before byPage-McCaw et al. ((2007) Nat Rev Mol Cell Biol 8(3):221-233). Enzymeinhibition studies were carried out using a Cary Eclipse fluorescencespectrophotometer (Varian, Walnut Creek, Calif., USA). Compound 1 wasstable in the buffers used in the kinetic assays.

Animals.

Female diabetic db/db mice (BKS.Cg-Dock7^(m)+/+Lepr^(db)/J, 8-weeks old)were purchased from the Jackson Laboratory (Bar Harbor, Me., USA) andfed 5001 Laboratory Rodent Diet (LabDiet, St. Louis, Mo., USA) and waterad libitum. Mice were housed in polycarbonate shoebox cages withhardwood bedding at 72±2° F. and 12 h light/12 h dark.

Excisional Diabetic Wound Model.

The dorsal area of the mice was shaved, and a single excisional 8-mmdiameter wound (50 mm²) was made on the dorsal thorax with a biopsypunch (Miltex, Plainsboro, N.J., USA) while the animals were underisofluorane anesthesia. Wounds were covered with Tegaderm™ dressing (3MCompany, St. Paul, Minn., USA). Topical treatment was started the nextday.

Wound Measurements.

Mice were anesthetized with isofluorane, and wounds were photographedwith an Olympus SP-800 UZ camera mounted on a tripod at a fixeddistance; a ruler was included in the digital photo. Wound areas werecalculated using NIH ImageJ software (version 1.48) and expressed aspercent change in wound area relative to day 0.

ND-336 Diabetic Wound Healing Study.

For this study, db/db mice were divided into two groups (n=8 mice pergroup): vehicle (50 μL of 20% DMSO, 80% propylene glycol per wound perday) and ND-336 (50 μL of a 2 mg/mL solution of ND-336 in 20% DMSO/80%propylene glycol, equivalent to 0.1 mg/wound/day). Wound measurementswere taken on days 0, 7, 10, and 14. Animals were sacrificed on day 14and wounds analyzed by H&E staining, TUNEL, and in-situ zymography.

Exogenous MMP-8 Study.

Female diabetic db/db (BKS.Cg-Dock7m+/+Leprdb/J, 8-weeks old, 38±3 g,n=24) were used for this study. Wounds were inflicted as described andthe following day the wounds were treated topically with MMP-8 (50 μL of20 μg/mL MMP-8 in reaction buffer) or vehicle (50 μL reaction buffer)once a day for 14 days. The reaction buffer consisted of 50 mM Tris (pH7.5), 10 mM CaCl₂, 150 mM NaCl, and 0.05% (w/v) Brij-35(polyoxyethyleneglycol dodecyl ether). Digital photographs of the woundswere taken on days 0, 7, 10, and 14 while animals were under isofluraneanesthesia. On days 7 and 14, 12 mice (n=6 per group) were sacrificed.The wounds were excised, embedded in OCT compound, and cryosectioned forhistological evaluation and in-situ zymography.

Combined ND-336 and MMP-8 Study.

Female db/db mice (n=6 per group) were divided into four groups: vehicle(50 μL of 10% DMSO/10% propylene glycol/80% saline per wound per daydosed in the morning and 50 μL of reaction buffer (50 mM Tris (pH 7.5),10 mM CaCl₂, 150 mM NaCl, and 0.05% (w/v) Brij-35) per wound per daydosed in the afternoon), ND-336 (50 μL of 1 mg/mL ND-336 (equivalent to0.05 mg/wound/day) in 10% DMSO/10% propylene glycol/80% saline dosed inthe morning and 50 μL of reaction buffer dosed in the afternoon), MMP-8(50 μL of 10% DMSO/10% propylene glycol/80% saline per wound per daydosed in the morning and 50 μL of 20 μg/mL of MMP-8 in reaction buffer(equivalent to 1 μg/wound/day) dosed in the afternoon), and combinedND-336 and MMP-8 (0.05 mg of ND-336 in 50 μL of 10% DMSO/10% propyleneglycol/80% saline per wound per day dosed in the morning and 1 μg ofMMP-8 in 50 μL of reaction buffer per wound per day dosed in theafternoon). Mice were sacrificed on days 7, 10, and 14, and the excisedwounds were embedded in OCT compound and cryosectioned for histologicalevaluation and in situ zymography.

Measurement of IL-6, TGF-β1, and VEGF by ELISA.

Wound tissues (n=3 mice/group) were harvested and immediately frozen inliquid nitrogen on days 1, 3, and 14. The extracted tissues werehomogenized in cold lysis buffer containing free-EDTA protease inhibitorcocktail (Pierce, Rockford, Ill., USA). The lysates were analyzed forprotein concentration by the Bradford protein assay (Bio-Rad, Hercules,Calif., USA). The levels of IL-6, TGF-β1 and VEGF in the lysates weredetermined by ELISA, following the manufacturer's protocol (Abcam,Cambridge, Mass., USA). The cytokine levels for each mouse sample wereexpressed in picograms/mg tissue.

Statistical Analyses.

Data were analyzed for statistical significance using the Student t-test(Excel) using a two-tail distribution and unequal variance.

MMP-9 Knockout Study.

Female MMP-9 knockout mice (B6.FVB(Cg)-Mmp9^(tm1Tvu)/J, 8-weeks old,19±2 g, n=14) and wild-type mice (C₅₇BLKS/6J, 8-weeks old, 19±2 g, n=14,same background as MMP-9 knockout mice) were used. The mice wereacclimated to the study room for one week prior to commencement of thestudy. Diabetes was induced by intraperitoneal injection ofstreptozotocin (Sigma-Aldrich, St. Louis, Mo.) at 150 mg/kg.Streptozotocin was dissolved in 100 mM sodium citrate buffer (pH 4.5)and administered within 15 min after preparation. After streptozotocintreatment, the mice were housed in disposable cages and given 10%sucrose water to drink for two days. The fasting blood glucose levelswere determined two days after streptozotocin treatment. Animals withblood glucose greater than 300 mg/dL were considered diabetic. Animalswith blood glucose less than 300 mg/dL received a second dose ofstreptozotocin one week later. The average blood glucose level of allthe animals was determined to be 465±113 mg/dL. Wounds were inflicted asdescribed and digital photographs were taken on days 0, 7, and 14. Mice(n=7 per group) were sacrificed on days 7 and 14, the wounds wereexcised, embedded in OCT compound, and cryosectioned for histologicalevaluation. FIG. 4 shows the effect of MMP-9 inhibition on diabeticwound healing. FIG. 5 shows the effect of MMP-9 gene ablation ondiabetic wound healing.

Cloning and Purification of Mouse MMP-8 and Effect of MMP-8 on DiabeticWound Healing.

The gene for the catalytic domain (304-852 bp), without the pro-domain,of MMP-8 was optimized for expression in Escherichia coli and wassynthesized by GenScript (Piscataway, N.J.) with unique NdeI and XhoIrestriction sites flanking the gene at the 5′ and 3′ termini,respectively. The gene was cloned into vector pET28a. E. coli DH5α wastransformed by this construct. The recombinant MMP-8 was expressed in E.coli BL21 (DE3) and purified using a previously published method (Botoset al. (1999) J Mol Biol 292(4):837-844), with induction by 0.5 mMisopropyl β-D-1-thiogalactopyranoside at 20° C. The purity of theprotein was determined to be >95% by SDS-PAGE. The enzyme concentrationwas evaluated spectrophotometrically using the extinction coefficientpredicted by ProtParam (Gasteiger et al. (2005) The Proteomics ProtocolsHandbook, ed. Walker J M (Humana Press), pp 571-607) (Δε₂₈₀=19681.6 M⁻¹cm⁻¹). Aliquots of the concentrated protein were stored in 50 mM Tris(pH 7.5), 5 mM CaCl₂, 300 mM NaCl, 20 μM ZnCl₂, 0.5% (w/v) Brij-35, 30%glycerol at −80° C. FIG. 6 shows that topical treatment with exogenouslyadded MMP-8 accelerates wound healing in db/db mice.

Dose-Response Study with ND-336.

We observed that ND-336 at a dose of 0.1 mg/wound/day accelerateddiabetic wound healing. In order to find the lowest dose of ND-336 thatwas efficacious in diabetic wound healing, a dose response study wasconducted at 0.05, 0.025, and 0.01 mg of ND-336/wound/day. For thisstudy, db/db mice were divided into four groups: vehicle (50 μL of 20%DMSO/80% propylene glycol, n=7 mice), 0.05 mg/wound/day ND-336 (50 μL of1 mg/mL ND-336 in 20% DMSO/80% propylene glycol, n=6 mice), 0.025mg/wound/day ND-336 (50 μL of 0.5 mg/mL ND-336 in 20% DMSO/80% propyleneglycol, n=7 mice), 0.01 mg/wound/day ND-336 (50 μL of 0.2 mg/mL ND-336in 20% DMSO/80% propylene glycol, n=7 mice).

As shown in FIG. 7, on day 14 the 0.05 mg/wound/day group exhibitedsignificant better healing than the vehicle and the 0.025 mg/wound/dayand 0.01 mg/wound/day groups. All the subsequent studies were performedat the 0.05 mg/wound/day level.

Histological Evaluation, Apoptosis Detection and In-Situ Zymography.

Wounds were embedded in optimal cutting temperature (OCT, Tissue-Tek,Sakura Finetek, Torrance, Calif., USA) compound and cryosectioned at12-μm thickness for hematoxylin-eosin (H&E) and at 8-μm thickness forin-situ zymography. Re-epithelialization was assessed on a fluorescentmicroscope (Nikon Eclipse 90i, Nikon Instruments, Inc., Melville, N.Y.,USA). Apoptotic cells in wound sections were assessed using a modifiedterminal deoxynucleotidyl transferase-mediated dUTP-nick-end labeling(TUNEL) assay kit, following the manufacturer's instructions (Trevigen,Inc., Gaithersburg, Md., USA). For in-situ zymography, unfixed cryostatsections of wound tissues were incubated in reaction buffer (50 mM TBSpH 7.6) containing DQ-gelatin or DQ-collagen (Molecular Probes, Inc.,Eugene, Oreg., USA) at 37° C. for 6 h. After fixation in 4%paraformaldehyde in PBS, cells were counterstained with DAPI and theimages were visualized by fluorescence microscopy. Immunofluorescentdetection of vascular density was performed by staining the tissues withAlexa Fluor 488 anti-mouse CD31 (BioLegend, San Diego, Calif., USA),followed by staining the nuclei with DAPI. The images for in-situzymography and vascular density were obtained by confocal microscopy.FIG. 8 shows that MMP-9 inhibition, exogenous MMP-8 treatment, andcombined MMP-9 inhibition and exogenous MMP-8 increase angiogenesis asmeasured by anti-CD31.

Example 3. Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a compound, enzyme, or composition described herein,or a pharmaceutically acceptable salt, solvate, or composition thereof(hereinafter referred to as ‘Compound X’):

(i) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/mL) mg/mL ‘Compound X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL ‘Compound X’ (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 0.1N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Compound X’ 20 Oleic acid 10Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

(vii) Topical Gel 1 wt. % ‘Compound X’   5% Carbomer 934 1.25%Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben  0.2%Purified water q.s. to 100 g

(viii) Topical Gel 2 wt. % ‘Compound X’    5% Methylcellulose    2%Methyl paraben  0.2% Propyl paraben 0.02% Purified water q.s. to 100 g

(ix) Topical Ointment wt. % ‘Compound X’ 5% Propylene glycol 1%Anhydrous ointment base 40%  Polysorbate 80 2% Methyl paraben 0.2%  Purified water q.s. to 100 g

(x) Topical Cream 1 wt. % ‘Compound X’  5% White bees wax 10% Liquidparaffin 30% Benzyl alcohol  5% Purified water q.s. to 100 g

(xi) Topical Cream 2 wt. % ‘Compound X’ 5% Stearic acid 10%  Glycerylmonostearate 3% Polyoxyethylene stearyl ether 3% Sorbitol 5% Isopropylpalmitate 2% Methyl Paraben 0.2%   Purified water q.s. to 100g

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient(s) ‘Compound X’. Aerosol formulation (vi) may be usedin conjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

1. A therapeutic composition for treating a wound comprising aneffective amount of the enzyme MMP-8 and an effective amount of aselective MMP-9 inhibitor.
 2. The composition of claim 1 wherein theselective MMP-9 inhibitor is selective for MMP-9 over MMP-8, wherein theKi value of the inhibitor for MMP-9 is at least 0.5 μM smaller than theKi value of the inhibitor for MMP-8.
 3. The composition of claim 1wherein the selective MMP-9 inhibitor is selective for MMP-9 over MMP-8,wherein the Ki value of the inhibitor for MMP-9 is at least 2 μM smallerthan the Ki value of the inhibitor for MMP-8.
 4. The composition ofclaim 1 wherein the MMP-9 inhibitor is:

or salt or solvate thereof.
 5. The composition of claim 1 wherein theMMP-9 inhibitor is:

or salt or solvate thereof.
 6. The composition of claim 3 wherein theMMP-9 inhibitor is:

or salt or solvate thereof.
 7. The composition of claim 6 wherein theMMP-9 inhibitor is:

or salt or solvate thereof.
 8. A therapeutic composition for treating awound comprising an effective amount of a recombinant MMP-8 enzyme incombination with a surfactant or buffer.
 9. The composition of claim 1in combination with a pharmaceutically acceptable diluent, excipient, orcarrier.
 10. A method of accelerating the healing of a wound comprisingcontacting an open wound with an effective amount of a compositioncomprising the enzyme MMP-8, thereby accelerating the healing of thewound.
 11. The method of claim 10 wherein the wound is contacted with atleast about 0.5 μg of MMP-8 per 50 mm² of open wound per day.
 12. Themethod of claim 10 further comprising administering to a subject havingthe open wound an effective amount of a selective MMP-9 inhibitor. 13.The method of claim 12 wherein the selective MMP-9 inhibitor isselective for MMP-9 over MMP-8, wherein the Ki value of the inhibitorfor MMP-9 is at least 0.5 μM smaller than the Ki value of the inhibitorfor MMP-8.
 14. The method of claim 13 wherein the MMP-9 inhibitor is:

or salt or solvate thereof.
 15. The method of claim 12 wherein theselective MMP-9 inhibitor is selective for MMP-9 over MMP-8, wherein theKi value of the inhibitor for MMP-9 is at least 2 μM smaller than the Kivalue of the inhibitor for MMP-8.
 16. The method of claim 15 wherein theMMP-9 inhibitor is:

or salt or solvate thereof.
 17. The method of claim 16 wherein the MMP-9inhibitor is:

or salt or solvate thereof.
 18. The method of claim 12 wherein at leastabout 0.05 mg of the MMP-9 inhibitor is administered to the subject per50 mm² of open wound per day.
 19. The method of claim 12 wherein thewound is a chronic wound and the subject is diabetic.
 20. A method fordecreasing inflammation and increasing angiogenesis in a diabetic woundcomprising administering to a subject having a diabetic wound aneffective amount of a composition of claim
 9. 21. A method for reducingthe amount of apoptotic cells in a diabetic wound comprisingadministering to a subject having a diabetic wound an effective amountof a composition of claim
 9. 22. (canceled)
 23. (canceled) 24.(canceled)